Although advances in the understanding of procaryotic organisms, particularly bacteria, having for the most part proceeded independently of advances in the understanding of eucaryotic organisms, it may be helpful to an appreciation of the present invention to set forth certain developments involving procaryotes.
In 1944, Avery reported the transformation of a procaryotic cell using DNA-mediated transfer of a cellular gene. Avery, O. T., et al., J. Exp. Med. 79: 137-158 (1944). Thereafter, reports of procaryotic transformation occurred in the literature. In 1975, Cohen and others reported results involving first transformation, then cotransformation of the procaryote Escherichia coli. Kretschmer, P. J., et al., J. Bacteriology 124: 225-231 (1975). In the experiments reported therein the authors disclosed the cotransformation of procaryotic cells using plasmid DNA, that is, extrachromosomal DNA which occurs naturally in many strains of Enterobacteriacae. In these experiments it was found that particular cells in a CaCl.sub.2 -treated bacterial population are preferentially competent for transformation. However, the frequency of transformation and the stability of the transformants obtained was low, possibly because the plasmid is not incorporated into the chromosomal DNA. As a result, cotransformants lost acquired traits after several generations. In addition, these experiments with bacteria required the addition of a gene promoter to the transforming DNA in order to obtain expression.
Meanwhile, experiments with eucaryotic cells proceeded substantially independently of those with procaryotic cells. In 1962, Szybalska, E. H. and Szybalski, W. PNAS 48: 2026 (1962) reported the transformation of mammalian cells but with such low frequency of transformation that it was not possible to distinguish transformants from cells which had merely undergone spontaneous reversion. Again, as with procaryotic cells, further reports of eucaryotic transformation occurred in the literature, but such results were oftentimes not reproducible by others. In addition, low frequencies of transformation, lack of understanding of the molecular basis for gene expression and the lack of molecular hybridization probes contributed to the lack of progress in this area. As a result, studies on the transformation of eucaryotic cells were essentially restricted to viral genes. Graham, F. L., et al., Cold Spring Harbor Symp. Quant. Biol. 39: 637-650 (1975) and McCutchen, J. H. and Pagano, J. S., Journal National Cancer Institute, 41: 351-357 (1968).
More recently, however, eucaryotic cells, specifically mammalian cells, were transformed with foreign DNA coding for a selectable phenotype. Wigler, M., et al., Cell 11: 223-232 (1977). This work has been extended and has resulted in the present invention wherein it has been discovered inter alia that eucaryotic cells can be cotransformed to yield transformants having foreign DNA integrated into the chromosomal DNA of the eucaryotic cell nucleus. Moreover, it has unexpectedly been discovered that such foreign DNA can be expressed by the cotransformants to generate functional proteins. In addition, by contrast with procaryotic transformants, the foreign DNA is stably expressed through hundreds of generations, a result that may be attributable to integration of the foreign DNA into the chromosomal DNA.
The present invention provides major advances over bacterial systems for future use in the commercial preparation of proteinaceous materials particularly proteins of eucaryotic origin such as interferon protein, antibodies, insulin, and the like. Such advantages include the ability to use unaltered genes coding for precursors for such proteinaceous materials. After cellular synthesis, the precursor can be further processed or converted within the eucaryotic cell to produce the desired molecules of biological significance. This phenomenon is well known for insulin which is initially produced in the eucaryotic cell as preproinsulin which is then converted to active insulin within the cell by appropriate peptide cleavage. Since procaryotic cells lack the requisite cellular machinery for converting preproinsulin to insulin, the insertion into a procaryotic cell of the eucaryotic gene associated with insulin will result in the production of preproinsulin, not insulin. Although, in the case of insulin, a relatively small and well characterized protein, this difficulty can be overcome by chemical synthesis of the appropriate gene, such an approach is inherently limited by the level of understanding of the amino acid sequence of the desired protein. Thus, for interferon protein, clotting factors, antibodies and uncharacterized enzymes, for which the exact amino acid sequence is not yet known, a procaryotic system will likely not prove satisfactory. By contrast, a eucaryotic system is not associated with such disadvantages since the eucaryotic cell possesses the necessary processing machinery. It is thus one important object of the present invention to provide a process for producing desired proteinaceous materials such as interferon protein, insulin, antibodies and the like which does not require a detailed molecular understanding of amino acid sequence.
In addition to the problem of precursors having additional amino acids which must be removed to produce active protein, important biological materials may be modified by chemical additions after synthesis and cleavage. Thus, for example, human-produced interferon is a glycoprotein containing sugar molecules in addition to protein. If produced in a bacterial cell, the interferon lacks the sugar molecules which are added when interferon is produced in a human cell. Moreover, proteinaceous materials produced within bacteria may include endotoxins which can cause inflammation if the proteinaceous material is administered to a mammal without significant purification. By contrast, interferon produced in a eucaryotic cell would be free of endotoxins.
It is therefore another important object of this invention to provide a process for producing compounds which include both non-proteinaceous and proteinaceous moieties such as glycoproteins which cannot be produced in bacterial cell.